The basic principle of infection prevention by vaccine is based on artificial pseudo-infection to induce acquired immunity, and to induce cellular immunity and antibody production against a specific pathogen. In acquired immunity, T cells and B cells in charge of immune “memory” play a central role, and variability of the variable region of the antibodies due to recombination of DNA is known to make possible an immune response having specificity for numerous antigens. On the other hand, in innate immunity, in which phagocytic cells such as leucocytes, macrophages, dendritic cells and the like play a central role is previously thought to perform non-specific phagocytic processing of pathogen and foreign objects, and to act only as a “temporary fix” until the establishment of acquired immunity. However, advances in research relating to the molecular mechanism of innate immunity reveals that specific recognition of self-versus-non-self clearly occurs, and innate immunity is indispensable for the establishment of acquired immunity. More specifically, recent research makes clear that the Toll-like receptor (TLR) family present in antigen-presenting cells, such as dendritic cells, macrophages, B cells and the like, induces acquired immunity through a reaction with various types of pathogens, induction of the production of cytokine, promotion of the differentiation of naïve T cells into Th1 cells, activation of killer T cells and the like.
One of a wide range of constituent components of pathogens recognized by a series of TLR family is a DNA having a CpG sequence (CpG DNA), which is a TLR9 ligand. The CpG sequence is a sequence of six bases having cysteine (C) and guanine (G) adjacent to one another at the central part of the sequence, and this base sequence is seldom found in mammals, although often seen in microorganisms. Moreover, most of the infrequently occurring CpG sequences in mammals are methylated. The non-methylated CpG sequences, which hardly exist in mammals, have strong immunostimulatory activity (for example, see Non-Patent Literature 1 to 3). CpG DNA imported into the cell by endocytosis is recognized by TLR9 present in the phagosome-like endoplasmic reticulum and strongly induces the Th1 reaction. In addition to suppressing the allergic reaction dominated by the Th2 reaction, the Th1 reaction has strong anti-tumorigenic activity. Thus in addition to protecting against infection, CpG DNA is anticipated to act as an adjuvant with respect to allergies and neoplastic disorders (for example, see Non-Patent Literature 4).
However, attempting to use CpG DNA as an immune therapy adjuvant leads to the problem of how to get the CpG DNA to arrive within the target cell while avoiding decomposition by nucleases in the cytoplasm and blood plasma and non-specific bonding with proteins.
The present inventors have paid attention to a polysaccharide having a β-1,3-glucan backbone (abbreviated hereinafter as “β-1,3-glucan”) as a novel gene carrier and discovered that β-1,3-glucan forms a new type of complex with various nucleic acids including nucleic acid medicines (anti-sense DNA, CpG DNA) (for example, see Patent Literature 1 and 2, and Non-Patent Literature 5-7).
The formation of a triple-strand helix complex by two β-1,3-glucan molecules and a single nucleic acid molecule is found to take place after dissolving β-1,3-glucan, which adopts a triple-strand helix structure in nature, in an aprotic polar organic solvent such as dimethyl sulfoxide (DMSO) and the like or in a 0.1N or stronger alkaline solution, to cause disassociation of the triple-strand helix into a single strand, adding a single-stranded nucleic acid, and switching the solvent to water or bringing the pH of the alkaline solution to neutral. In this case, β-1,3-glucan molecules and the nucleic acid molecule in the triple-strand helix complex are considered to form intermolecular bonding mainly through hydrogen bonds and hydrophobic interaction.
By complexation of the nucleic acid and β-1,3-glucan in the above described manner, the nucleic acid may be delivered into the cell while suppressing hydrolysis of the nucleic acid molecule by nucleases in the cytoplasm, and while suppressing undesirable interactions between the nucleic acid molecule and proteins in the body such as non-specific bonding of the nucleic acid molecules with proteins in the blood plasma. CpG DNA is successfully delivered into the cell by using the complex of β-1,3-glucan and nucleic acid, and by using a ternary complex further containing an antigenic peptide, as seen, for example, in Patent Literature 3 and 4, and in Non-Patent Literature 9 to 11.